Sterilizing polymer and method of preparation thereof

ABSTRACT

The present invention provided a polymer and a method for preparing the polymer, wherein the polymer was a terpolymer having a molecular weight of more than 1000, and copolymerized by acrolein, acrylic acid, and low alkene-alkyl dibasic acid or low-alkene-alkyl polybasic acid.

FIELD OF INVENTION

The invention provides an antimicrobial polymer and a method for preparing the polymer, wherein the polymer is a terpolymer having a molecular weight of more than 1000, and copolymerized by acrolein, acrylic acid, and low alkene-alkyl dibasic acid or low-alkene-alkyl polybasic acid.

BACKGROUND OF THE INVENTION

Research on the acrolin polymer was first seen in patent CN87108390 applied by Melrose Graham (Australia) in 1988. The patent introduced polyacrolein polymer with antimicrobial activities. The formation of polymer could eliminate the toxicity of low molecular weight and pungent odor of acrolin, while retaining the biological activity of acrolein. However, due to the extremely poor dissolubility, the application of polyacrolein was limited. In patent CN1875039, the method of heating and adding polyethylene glycol was used to promote the dissolubility of polyacrolein, but the dissolution was very time consuming and with poor stability. In patent AU-A-11686/95, it was mentioned that hydrogen peroxide could be used to oxidize partial acetal radical into carboxyl group to improve the stability. In patent CN1291895, acrylic acid and acrolein were copolymerized to improve the dissolubility, but the dissolubility would be decreased with the increase in the proportion of acrolein. In this patent, it was also mentioned to use surfactant and acidifier to improve the chemical stability of the polymer. In patent CN1347425 and CN1617733, polyoxyl, alcohol phenyl, polyalcohol and/or alkanol were added to improve the stability and bactericidal activity of poly (acrolein, acrylic acid). However, it had been found in some studies that, the biological activities were not improved when polyoxyl, alcohol phenyl, polyalcohol and/or alkanol were added. The original defect still existed, and this type of the polymers including the following repeating units:

Wherein R is H, n is an integer≧1.

SUMMARY OF THE INVENTION

The objective of this invention is to provide a new polymer and a preparing method to overcome the above defects.

In one embodiment, the invention provides a bactericidal polymer, which is a terpolymer having a molecular weight of 1000-10000 Daltons, and formed by copolymerizing of acrolein, acrylic acid, and low molecular weight alkene-alkyl dibasic acid or polybasic acid. Its structure comprises one or more repeating units. Examples of repeating units include the following structures A-E:

Wherein m and n are integers≧1, R is H or —R₁COOH, and R₁ is a C₀-C₁₀ alkyl group, alkenyl or polybasic alkyl group with double bonds. In another embodiment, R₁ could be C₀-C₅ alkyl group. In another embodiment, R₁ could be C₀-C₂ alkyl group, wherein R=(—CH₂COOH).

In one embodiment, the polymer content of carboxyl group is 0.15-7.5 mole/kg and molecular weight of the polymer is 1000-5000.

In one embodiment, the raw materials include: acrolein, acrylic acid and alkenyl polybasic acid, wherein the polybasic acid can be maleic acid, fumaric acid, trans-glutaconic acid or cis-glutaconic acid.

In one embodiment, the raw materials of this invention include acrylic acid, acrolein and cis (trans) maleic acid. In another embodiment, raw materials are acrylic acid, acrolein and fumaric acid. In one embodiment, the mole ratio range of acrylic acid:acrolein:fumaric acid is 1-100:1-100:1-100. In another embodiment, acrylic acid:acrolein:fumaric acid is 1-98: 1-50:1-50. In another embodiment, acrylic acid:acrolein:fumaric acid is 40-90: 10-50:1-10. In another embodiment, acrylic acid:acrolein:fumaric acid is 50-70: 20-40:5-9, and in still yet embodiment, acrylic acid:acrolein:fumaric acid is 63:30:7.

The polymer of this invention can be prepared through the following steps:

Distilled acrylic acid, distilled acrolein, and fumaric acid were added into a three-port flask with absolute methanol and purged with nitrogen, benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen. The reacting solution became a brown viscous liquid, which was dried under vacuum to form acrylic acid-acrolein-fumaric acid copolymer.

The invention also includes the pharmaceutical compositions containing the invented polymer as the active ingredients and the pharmaceutically acceptable carrier. The polymer of this invention can be combined with other active ingredients as the synergist of antibacterial and anti-coccidiosis, as long as they are not antagonistic.

The pharmaceutical compositions of this invention can be formulated into any suitable dosage form, e.g.: oral preparation (such as tablet, capsules, oral liquid, suspension, pills and granules), local agents (such as ointment, solution and suppositories, etc). The pharmaceutical compositions of this invention can be prepared in accordance with the well known method in pharmaceutical field.

The carriers of pharmaceutical compositions in this invention are easily available in pharmaceutical field, including adhesives, lubricants, disintegrant, dispersion agents, solvents, diluents, stabilizers, suspending agents, flavoring agents etc., which are used in oral preparations and controlled release carriers for sustained-release or controlled release preparations; as well as matrix, thinners, lubricants, preservatives for local agents, etc.

Another objective of this invention is to provide this polymer in the preparations against bacterial and parasitic infections in animals and humans.

The dose of polymer in this invention can be properly adjusted according to therapeutic efficacy of in vivo bioavailability, metabolism and excretion rate of active ingredients, the age, sex, and disease stage of the patients. Therefore, the therapeutic dose can be a wide range of changes. Generally, the dose of the polymer can be adjusted according to the actual amounts of therapeutic ingredients or pharmaceutical compositions mentioned in this invention to reach the request of effective treatment, and complete the purpose of this invention to treat the infections. Effective dose can follow the guidance of a doctor or physician, as a single dose administration or in the form of several certain intervals or continuously administration.

The doses of the above active ingredients can be changed according to different formulae. However, if necessary, the above mentioned doses may be deviated, which is determined by the category and bodyweight of the subjects, individual pharmaceutical behavior, nature and severity of the disease, type of preparation and method of administration, as well as time and intervals of administration.

The invention increases the proportion of carboxy groups in the polymer to promote its dissolubility. Meanwhile, it has disclosed stronger bactericidal activity in vitro.

The following contrast experiments illustrate the beneficial effects of the invention:

I. Synthesis Experiments

Synthesis of control drug (mole ratio, acrolein: acrylic acid=70:30): 24.55 g freshly distilled acrolein and 72.92 g freshly distilled acrylic acid were added into a three-port flask with 210 ml absolute methanol, purged with nitrogen, 4.41 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 70 h. The reacting solution became a brown viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form poly (acrolein-acrylic acid) named A1.

Synthesis of the invented drug (mole ratio, acrolein:acrylic acid:fumaric acid=63:30:7): 73.7 g freshly distilled acrolein, 98.8 g freshly distilled acrylic acid and 35.6 g fumaric acid were added into a three-port flask with 630 ml absolute methanol, purged with nitrogen, and 13.2 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 70 h. The reacting solution became a brown viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form poly (acrolein, acrylic acid, fumaric acid) named A2.

II. Contrast Experiments of Dissolubility:

Weighed 2.40 g Poly (acrolein-acrylic acid) A1 and 2.4 g Poly (acrolein, acrylic acid, fumaric acid) A2 as abovementioned were added into 100 ml water respectively, heated to boiling, then observed the phenomena and determined pH values; then added sodium carbonate to adjust the solution to be transparent, and adjusted pH value to be acidic and alkaline groups, the corresponding acidic solutions were B1 and B2, and the alkaline solutions were C1 and C2. The dissolving results were shown in table 1.

TABLE 1 boiling without added sodium carbonate sodium carbonate Adjust Adjust Name Phenomena PH Phenomena PH to PH to Poly A1 Only Turbid 4.10 8.49 (C1) (acrolein, swelling solution (B1) acrylic after only after acid) boiling for boiling for 24 h more than 20 h, Poly A2 milky 2.25 transparent 4.15 8.46 (C2) (acrolein, solution solution (B2) acrylic after acid, boiling for fumaric 30 min acid)

III. Contrast Experiments of Toxicity:

Experimental method: bodyweight 18-22 g white rats, half ♀ and half ♂, fasted for 12 h before the experiment, and water was provided freely. Ten white rats were divided into one group. Made polymer A1 and A2 into a 50% solution, and injected 5000 mg/kg bodyweight and 10000 mg/kg bodyweight through the white rats mouth into their stomachs separately. White rats in polymer A2 group injected 10000 mg/kg all died, so 6500 mg/kg bodyweight and 8000 mg/kg bodyweight were additionally administered. The white rats were observed for 14 days after administration, and the experimental results were shown in table 2.

TABLE 2 Dose (mg/kg Dead number of polymer name body weight) white rats Poly (acrolein, 5000 1 acrylic acid) A1 10000 3 Poly (acrolein, 5000 1 acrylic acid, 6500 1 fumaric acid) 8000 3 A2 10000 10

IV. Contrast Experiments of Bactericidal Efficacy: Experimental Method 1. Bacteria Cultivation

Crossed the bacteria strains and inoculated onto MH solid culture medium then cultured for 24 h, picked single colony by a sterilized tip and inoculated into 3 ml MH broth culture medium, then placed into a 37° C. constant temperature incubator for 18 h.

2. Bacteria Count

Put 8 sterilized tubes (13×10 mm) on the tube shelf in order from 1 to 8. Added 9 ml sterilized MH broth medium in each tube, then added 1 ml of the above-mentioned bacteria solution into No. 1 tube, after mixing up, withdrew 1 ml and added into No. 2 test tube, then mixed up, withdrew 1 ml into No. 3 test tube, and diluted in order till the No. 8 test tube, took No. 5-8 test tube samples, withdrew 0.1 ml bacteria solution from each test tube and smeared onto the sterilized MH solid culture medium, and each sample was tested for 3 times. Placed the culture medium into a 37° C. constant temperature incubator and cultured for 18 hours. Chose appropriate culture dish to count the bacteria, and adopt mean value as the concentration of bacteria solution.

3. Determining Method

Preparation of Standard Bacteria Solution: Diluted the Cultured and Counted Fresh bacteria solution with MH broth culture medium for later use.

Assay: took 10 sterilized test tubes (13×10 mm) and put onto the tube shelf in order from No. 1 to 10. Added 1.8 ml the above diluted bacterial solution into No. 1 test tube, and withdrew 1 ml into No. 2-8 tubes. Added 0.2 ml polymer sample into No. 1 test tube, and mixed up. Withdrew 1 ml solution from No. 1 test tube into the No. 2 test tube, and diluted the bacteria solutions from No. 2 to No. 10 test tubes, and discard 1 ml from the No. 10 test tube. Stopped up all the test tubes, cultured in a 37° C. incubator for 18 h, and observed the results.

Staphylococcus aureus: Staphylococcus aureus was isolated from chicken intestines in chicken farm, bacteria concentration was 1.7×10⁸ cfu/ml, and the bactericidal efficacies were shown in table 3.

TABLE 3 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B1 + + − C1 + − − B2 + + − C2 + + − + efficient, − inefficient

2) Bacillus coli: Bacillus coli was isolated from chicken intestines in chicken farm, bacteria concentration was 5×10⁶ cfu/ml, and the bactericidal efficacies were shown in table 4.

TABLE 4 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B1 + + − C1 − − − B2 + + − C2 − − − + efficient, − inefficient

3) Bacillus aeruginosus: Bacillus aeruginosus was standard bacteria strain at a concentration of 3.1×10⁵ cfu/ml, and the bactericidal efficacies were shown in table 5.

TABLE 5 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B1 + + − C1 + − − B2 + + − C2 + − − + efficient, − inefficient

4) Salmonella: Salmonella was isolated from chicken intestines in chicken farm, bacteria concentration was 3.4×10⁴ cfu/ml, and the bactericidal efficacies were shown in table 6.

TABLE 6 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B1 + + − C1 + − − B2 + + − C2 + + − + efficient, − inefficient

Experimental Conclusion:

As observed in table 1, the dissolubility of A2 was better than that of A1, which meant that biprotic acid was introduced in poly (acrolein, acrylic acid) greatly improved dissolubility of the polymer, and the stability of the polymer solution was also improved.

As observed in table 2, 3 rats died at dose of 10000 mg/kg bodyweight poly (acrolein, acrylic acid), so we inferred that the LD50 of poly (acrolein, acrylic acid) was no less than 10000 mg/kg bodyweight; 3 rats died at the dose of 8000 mg/kg bodyweight poly (acrolein, acrylic acid, fumaric acid), so its LD50 was between 8000 and 10000 mg/kg bodyweight. The toxicity of poly (acrolein, acrylic acid, fumaric acid) was higher than that of poly (acrolein, acrylic acid), but both of the polymers are all Low toxicity.

As observed in table 3, 4, 5 and 6, the results of bactericidal effects of the two polymers on Staphylococcus aureus, Bacillus coli, Bacillus aeruginosus and Salmonella indicated that the bactericidal effects of poly (acrolein, acrylic acid) and poly (acrolein, acrylic acid, fumaric acid) were equivalent under acidic conditions. However, in alkaline conditions, the bactericidal effects of poly (acrolein, acrylic acid, fumaric acid) were superior to that of poly (acrolein, acrylic acid). Moreover, the bactericidal effects of any polymer under acidic conditions were better than that under alkaline conditions.

Therapeutic experiments of the polymer of this invention to colibacillosis by artificial induction in broiler.

Test materials: The polymer of this invention was provided by Qingdao Continent Pharmaceutical Co., Ltd.; premixed origanum oil was bought from Weifang Jiajia Animal Husbandry R&D Co., Ltd., and Amikacin Sulfate was bought from Qilu Pharmaceutical Co., Ltd.

Test animals: 500 healthy AA⁺ chicks of 1 day old with similar primary bodyweight were chosen. The chicks were bred in the cages. Feeds were self-made powder, and the chicks could freely drink water and eat food under light for 24 h a day.

Infecting bacteria strain: Standard bacterial strain of Chick E. coli O₇₈ (CVCC4904) was bought from Control Institute of Veterinary Bioproducts and Pharmaceuticals, China.

Experiment: Animal groups: 350 healthy chicks of 21-day-old were randomized into 10 groups, with 35 chicks in each group.

Inoculation: 1 mL of E. coli solution was injected peritoneally for each chick to induce the disease. Drugs were administered when the chick fall ill.

TABLE 7 drug groups and dose Number Drugs pH value Dose Group 1 Polymer A2 7.18 500 PPM administered with drinking water, 7 days continuously, twice daily Group 2 Polymer A2 7.18 400 PPM administered with drinking water, 7 days continuously, twice daily Group 3 Polymer A2 7.18 300 PPM administered with drinking water, 7 days continuously, twice daily Group 4 Polymer A2 7.18 200 PPM administered with drinking water, 7 days continuously, twice daily Group 5 Polymer A1 7.11 400PPML administered with drinking water, 7 days continuously, twice daily Group 6 Amikacin 210 PPM (as Amikacin) sulfate administered with drinking water, 7 days continuously, twice daily Group 7 Amikacin 210 PPM (as Amikacin) + sulfate + 300 PPM administered with polymer A2 drinking water, 7 days continuously, twice daily Group 8 Origanum oil 100 PPM mixed with feeds, 7 days continuously, twice daily Group 9 Infection group without administration Group 10 Non infection group without administration

The chicks were inoculated by bacteria in test group when they were 24-day-old, different doses of drugs were added in drinking water after 12 h of inoculation for 7 days continuously, and twice daily.

Determined parameters: mortality, cured rate and effective rate of each drug group.

dead number: dead number was calculated by the number of died chicks which appeared typical symptoms of colibacillosis during the experiment and E. coli could be isolated from heart and liver of died chicks.

Cured number: Cured number was calculated based on the number of cured chicks which had recovered normal mental state after administration, with no decreased appetite, no dysentery etc.

Effective number: Effective number was calculated according to the number of living chick after the test including the cured or living chicks with clinical symptoms after administration.

Results: Comparison of treatment effects on E. coli of polymer A2, A1, amikacin sulfate, Amikacin Sulfate+polymer A2 and origanum oil were shown in table 8.

TABLE 8 Groups Remain Group Dead in Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 10 chicks chicks Drug name Polymer Polymer Polymer Polymer Polymer Amikacin Amikacin Origanum Infection Non A2 A2 A2 A2 A1 sulfate sulfate + cal group infec- PolymerA2 without tion admin- group istration without drug Chick num 35 35 35 35 35 35 35 35 35 35 350 Died number 1 3 1 3 1 4 1 4 0 0 18 332 during 0-12 h after inoculation Chick number of 34 32 34 32 34 31 34 31 35 35 332 administration Dead number 1 1 2 4 1 4 1 1 1 0 16 316 at day 1 Dead number 0 0 2 2 1 0 0 1 2 0 8 308 at day 2 Dead number 0 0 1 0 1 0 1 1 1 0 3 303 at day 3 Dead number 0 0 0 0 0 0 0 0 3 0 3 300 at day 4 Dead number 0 0 0 0 0 0 0 0 3 0 3 297 at day 5 Dead number 0 0 1 0 0 0 0 0 2 0 3 294 at day 6 Dead number 0 0 0 1 0 0 0 0 1 0 2 292 at day 7 Total dead 1 1 6 7 3 4 2 3 13 0 40 number during administration (25-32 days) Dead number after 0 0 1 0 3 2 0 3 2 0 11 281 withdrawal (33-42 days) Total dead 1 1 7 7 6 6 2 6 15 0 51 number after administration (26-42 days) remaining 33 31 27 25 28 25 32 25 20 35 — 281 chick number heating 28 25 20 16 25 18 26 17 11 35 — 221 chick number Sick chick 5 6 7 9 3 7 6 8 9 0 — 60 number Mortality (%) 2.94 3.13 20.59 21.88 17.65 19.35 5.88 19.35 42.86 0 — Cured rate (%) 82.35 78.13 58.82 50.00 73.53 58.06 76.47 54.84 31.43 0 — — (self cured) Effective rate (%) 97.08 96.87 79.41 78.12 82.35 80.65 94.12 80.85 — — — —

Note: mortality was Total dead number after administration/chick number before administration; cured rate was healthy chick number after administration/chick number before administration; effective rate was remaining chick number after administration/chick number before administration; Performance of sick chicks: depressed, loose and disordered feather, or pendulous wing, eyes unopened, poor appetite, emaciated, swollen articulation, not stand and weighed less than 1000 g.

Conclusion: as Shown in Table 8:

In table 8, group 2 and group 5 were the same dose of polymer A2 and polymer A1 and the results indicated that the mortality rates of group A2 and A1 were 3.13% and 17.65%, the cured rates were 78.13% and 73.53%, the effective rates were 96.87% and 82.35% respectively, and the effects of polymer A2 were superior to polymer A1.

In table 8, as observed in group 1, group 2, group 6 and group 8, when polymer A2 was administered at a dose more than 400 PPM, the mortality rate was far lower than that in groups administered 210 PPM amikacin sulfate and 100 PPM origanum oil, the cured rates and effective rates were also superior to those of amikacin sulfate and origanum oil groups, with significant difference; effects of polymer A2 in 300 PPM group were equivalent to that groups of amikacin sulfate in 210 PPM and origanum oil in 100 PPM, with no significant differences.

In table 8, comparison between group 6 and 7 indicated that polymer A2 had significant synergistic effects on amikacin sulfate in treating colibacillosis.

Implementation:

The following examples illustrated the invention but not limit the invention.

EXAMPLE 1

Control drug: (acrylic acid:acrolein (mol)=95:5): 3.25 g freshly distilled acrolein and 79.27 g freshly distilled acrylic acid were added into a three-port flask with 520 ml absolute methanol, purged with nitrogen, 4.74 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 50 h. The reaction solution became a yellowish viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form poly (acrolein-acrylic acid) named A3.

Invented drug (acrylic acid: acrolein: fumaric acid (mol)=90:5:5): 3.89 g freshly distilled acrolein, 90.08 g freshly distilled acrylic acid and 8.06 g fumaric acid were added into a three-port flask with 590 ml absolute methanol, purged with nitrogen, 5.04 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 50 h. The reaction solution became a yellowish viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form Poly (acrolein, acrylic acid, fumaric acid) named A4.

Contrast Experiments of Dissolubility:

The results were shown in table 9.

TABLE 9 Boiling without added sodium carbonate sodium carbonate Adjust Adjust Name Phenomena PH Phenomena PH to PH to Poly A3 Turbid 2.10 Transparent 4.06 8.26 (acrolein, solution solution (B3) (C3) acrylic acid) after boiling Poly A4 Basically 2.15 Transparent 4.10 8.24 (acrolein, transparent solution (B4) (C4) acrylic acid, solution after fumaric boiling acid)

Contrast Experiments of Toxicity:

The results were shown in table 10:

TABLE 10 Dose (mg/kg Number of Polymer name body weight) dead rats Poly (acrolein, acrylic acid) 5000 0 A3 10000 1 Poly (acrolein, acrylic acid, 5000 0 fumaric acid)A4 10000 1

Contrast Experiments of Bactericidal Efficacy:

1) Staphylococcus aureus: Staphylococcus aureus was isolated from chick intestines in the chicken farm, the concentration of bacteria was 2.5×10⁶ cfu/ml, and the bactericidal efficacies were shown in table 11.

TABLE 11 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B3 + + + − C3 + − − − B4 + + + − C4 + + − − + efficient, − inefficient

2) Bacillus coli: Bacillus coli was isolated from chick intestines in the chicken farm, the concentration of bacteria was 2×10⁸ cfu/ml, and the bactericidal efficacies were shown in table 12.

TABLE 12 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B3 + − − C3 − − − B4 + + − C4 − − − + efficient, − inefficient

3) Bacillus aeruginosus: Bacillus aeruginosus was standard bacteria strain at a concentration of 1.7×10⁵ cfu/ml, and the bactericidal efficacies were shown in table 13.

TABLE 13 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B3 + − − C3 − − − B4 + + − C4 − − − + efficient, − inefficient

4) Salmonella: Salmonella was isolated from chick intestines in the chicken farm, the concentration of bacteria was 1.9×10⁵ cfu/ml, and the bactericidal efficacies were shown in table 14.

TABLE 14 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B3 + − − C3 − − − B4 + + − C4 + − − + efficient, − inefficient

EXAMPLE 2

Control drug: (acrylic acid: acrolein (mol)=90:10): 7.04 g freshly distilled acrolein and 81.43 g freshly distilled acrylic acid were added into a three-port flask with 530 ml absolute methanol, purged with nitrogen, 5.47 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 50 h. The reaction solution became a yellowish viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form poly (acrolein-acrylic acid) named A5.

Invented drug (acrylic acid: acrolein: fumaric acid (mol)=85:10:5): 8.05 g freshly distilled acrolein, 87.91 g freshly distilled acrylic acid and 8.34 g fumaric acid were added into a three-port flask with 570 ml absolute methanol, purged with nitrogen, 5.18 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 50 h. The reaction solution became a yellowish viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form poly (acrolein, acrylic acid, fumaric acid) named A6.

Contrast Experiments of Dissolubility:

The results were shown in table 15.

TABLE 15 Boiling without added sodium carbonate sodium carbonate Adjust Adjust Name Phenomena PH Phenomena PH to PH to Poly A5 Turbid solution 2.15 Transparent 4.12 8.36 (acrolein, after boiling solution (B5) (C5) acrylic acid) Poly A6 Basically 2.18 Transparent 4.10 8.28 (acrolein, transparent solution (B6) (C6) acrylic solution after acid, boiling fumaric acid)

Contrast Experiments of Toxicity:

The results were shown in table 16:

TABLE 16 Polymer name Dose (mg/kg body weight) Number of dead rats Poly (acrolein, acrylic 5000 0 acid) A5 10000 1 Poly (acrolein, acrylic 5000 1 acid, fumaric acid) A6 10000 1

Contrast experiments of bactericidal efficacy:

1) Staphylococcus aureus: Staphylococcus aureus was isolated from chick intestines in the chicken farm, the concentration of bacteria was 5.0×10⁷ cfu/ml, and the bactericidal efficacies were shown in table 17.

TABLE 17 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B5 + − − − C5 − − − − B6 + + + − C6 + − − − + efficient, − inefficient

2) Bacillus coli: Bacillus coli was isolated from chick intestines in the chicken farm, the concentration of bacteria was 3.5×10⁷ cfu/ml, and the bactericidal efficacies were shown in table 18.

TABLE 18 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B5 + + − − C5 + − − − B6 + + + − C6 + − − − + efficient, − inefficient

3) Bacillus aeruginosus: Bacillus aeruginosus was standard bacteria strain at a concentration of 2.8×10⁵ cfu/ml, and the bactericidal efficacies were shown in table 19.

TABLE 19 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B5 + + − C5 − − − B6 + + − C6 + − − + efficient, − inefficient

4) Salmonella: Salmonellae was isolated from chick intestines in the chicken farm, the concentration of bacteria was 2.1×10⁵ cfu/ml, and the bactericidal efficacies were shown in table 20.

TABLE 20 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B5 + + − C5 + − − B6 + + − C6 + + − + efficient, − inefficient

EXAMPLE 3

Control drug: (acrylic acid:acrolein (mol)=80:20): 39.15 g freshly distilled acrolein and 201.24 g freshly distilled acrylic acid were added into a three-port flask with 600 ml absolute methanol, purged with nitrogen, 12.78 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 60 h. The reaction solution became a yellow viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form poly (acrolein, acrylic acid) named A7.

Invented drug (acrylic acid: acrolein: fumaric acid (mol)=72:20:8): 42.99 g freshly distilled acrolein, 198.9 g freshly distilled acrylic acid and 35.58 g fumaric acid were added into a three-port flask with 570 ml absolute methanol, purged with nitrogen, 11.73 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 60 h. The reaction solution became a yellow viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form Poly (acrolein, acrylic acid, fumaric acid) named A8.

Contrast Experiments of Dissolubility:

The results were shown in table 21.

TABLE 21 Boiling without added sodium carbonate sodium carbonate Adjust Adjust Name Phenomena PH Phenomena PH to PH to Poly A7 small jelly transparent 4.02 8.10 (acrolein, after boiling solution after (B7) (C7) acrylic acid) for 24 h boiling for 12 h Poly A8 Milky 2.16 Transparent 4.00 8.12 (acrolein, solution solution (B8) (C8) acrylic acid, after boiling fumaric acid) for 10 min

Contrast Experiments of Toxicity:

The results were shown in table 22:

TABLE 22 Polymer name Dose (mg/kg body weight) Number of dead rats Poly (acrolein, acrylic 5000 1 acid) A7 10000 2 Poly (acrolein, acrylic 5000 0 acid, fumaric acid) A8 10000 2

Contrast Experiments of Bactericidal Efficacy:

1) Staphylococcus aureus: Staphylococcus aureus was isolated from chick intestines in the chicken farm, the concentration of bacteria was 5.8×10⁶ cfu/ml, and the bactericidal efficacies were shown in table 23.

TABLE 23 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B7 + + − − C7 + − − − B8 + + + − C8 + − − − + efficient, − inefficient

2) Bacillus coli: Bacillus coli was isolated from chick intestines in the chicken farm, the concentration of bacteria was 2.0×10⁷ cfu/ml, and the bactericidal efficacies were shown in table 24.

TABLE 24 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B7 + − − − C7 + − − − B8 + + + − C8 + − − − + efficient, − inefficient

3) Bacillus aeruginosus: Bacillus aeruginosus was standard bacterial strain at a concentration of 2.0×10⁵ cfu/ml, and the bactericidal efficacies were shown in table 25.

TABLE 25 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B7 + − − − C7 − − − − B8 + + + − C8 + − − − + efficient, − inefficient

4) Salmonella: Salmonella was isolated from chick intestines in the chicken farm, the concentration of bacteria was 9.1×10⁴ cfu/ml, and the bactericidal efficacies were shown in table 26.

TABLE 26 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B7 + + − − C7 + − − − B8 + + + − C8 + + − − + efficient, − inefficient

EXAMPLE 4

Control drug: (acrylic acid: acrolein (mol)=75:25): 47.64 g freshly distilled acrolein and 183.75 g freshly distilled acrylic acid were added into a three-port flask with 540 ml absolute methanol, purged with nitrogen, 11.43 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 65 h. The reaction solution became a yellow viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form poly (acrolein, acrylic acid) named A9.

Invented drug (acrylic acid:acrolein:fumaric acid (mol)=65:25:10): 56.91 g freshly distilled acrolein, 190.23 g freshly distilled acrylic acid and 47.13 g fumaric acid were added into a three-port flask with 570 ml absolute methanol, purged with nitrogen, 12.30 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 65 h. The reaction solution became a yellow viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form Poly (acrolein, acrylic acid, fumaric acid) named A10.

Contrast Experiments of Dissolubility:

The results were shown in table 27.

TABLE 27 Boiling without added sodium carbonate sodium carbonate Adjust Name Phenomena PH Phenomena PH to Adjust PH to Poly (acrolein, A9 Only swelling transparent 4.22 (B9)  8.16 acrylic acid) after boiling for solution (C10) 24 h after boiling for 20 h Poly (acrolein, A10 Turbid solution 2.22 transparent 4.25 (B10) 8.18 acrylic acid, after boiling for sodium (C10) fumaric acid) 1 h

Contrast Experiments of Toxicity:

The results were shown in table 28:

TABLE 28 polymer name Dose (mg/kg body weight) Number of dead rats Poly (acrolein, acrylic 5000 1 acid) A9 10000 3 Poly (acrolein, acrylic 5000 1 acid, fumaric acid) 10000 4 A10

Contrast Experiment of Bactericidal Efficacy:

1) Staphylococcus aureus: Staphylococcus aureus was isolated from chick intestines in the chicken farm, the concentration of bacteria was 8.2×10⁶ cfu/ml, and the bactericidal efficacies were shown in table 29.

TABLE 29 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B9 + + + − C9 + − − − B10 + + + − C10 + + − − + efficient, − inefficient

2) Bacillus coli: Bacillus coli was isolated from chick intestines in the chicken farm, the concentration of bacteria was 8.4×10⁶ cfu/ml, and the bactericidal efficacies were shown in table 30.

TABLE 30 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B9 + − − C9 − − − B10 + + − C10 − − − + efficient, − inefficient

3) Bacillus aeruginosus: Bacillus aeruginosus was standard bacterial strain at a concentration of 1.3×10⁶ cfu/ml, and the bactericidal efficacies were shown in table 31.

TABLE 31 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B9 + − − C9 + − − B10 + + − C10 + − − + efficient, − inefficient

4) Salmonella: Salmonella was isolated from chick intestines in the chicken farm, the concentration of bacteria was 3.3×10⁵ cfu/ml, and the bactericidal efficacies were shown in table 32.

TABLE 32 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B9 + − − C9 − − − B10 + + − C10 + − − + efficient, − inefficient

EXAMPLE 5

Control drug (acrolein:acrylic acid (mol)=65:35): 69.72 g freshly distilled acrolein and 166.44 g freshly distilled acrylic acid were added into a three-port flask with 500 ml absolute methanol, purged with nitrogen, 10.98 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 75 h. The reaction solution became a dark brown viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form poly (acrolein, acrylic acid) named A11.

The invented drug (acrolein:acrylic acid:fumaric acid (mol)=55:35:10): 78.12 g freshly distilled acrolein, 150.80 g freshly distilled acrylic acid and 46.23 g fumaric acid were added into a three-port flask with 590 ml absolute methanol, purged with nitrogen, 9.48 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 75 h. The reaction solution became a dark brown viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form Poly (acrolein, acrylic acid, fumaric acid) named A12.

Contrast Experiment of Dissolubility:

The results were shown in table 33.

TABLE 33 Boiling without added sodium carbonate sodium carbonate Adjust Name Phenomena PH Phenomena PH to Adjust PH to Poly A11 Only swelling turbid solution 4.26 (B11) 8.30 (acrolein, after boiling after boiling for (C11) acrylic acid) for 24 h 48 h Poly A12 turbid solution 2.28 Transparent 4.18 (B12) 8.24 (acrolein, after boiling solution after (C12) acrylic acid, boiling for 2 fumaric hours acid)

Contrast Experiment of Toxicity:

The results were shown in table 34:

TABLE 34 Polymer name Dose (mg/kg body weight) Number of dead rats Poly (acrolein, acrylic 5000 1 acid) A11 6500 1 8000 4 10000 8 Poly (acrolein, acrylic 5000 1 acid, fumaric acid)A12 6500 2 8000 5 10000 10

Contrast Experiment of Bactericidal Efficacy: Experiment

1) Staphylococcus aureus: Staphylococcus aureus was isolated from chick intestines in the chicken farm, the concentration of bacteria was 3.2×10⁸ cfu/ml, and the bactericidal efficacies were shown in table 35.

TABLE 35 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B11 + + − − C11 + − − − B12 + + + − C12 + + − − + efficient, − inefficient

2) Bacillus coli: Bacillus coli was isolated from chick intestines in the chicken farm, the concentration of bacteria was 1.6×10⁷ cfu/ml, and the bactericidal efficacies were shown in table 36.

TABLE 36 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 B11 + − − C11 − − − B12 + + − C12 − − − + efficient, − inefficient

3) Bacillus aeruginosus: Bacillus aeruginosus was standard bacterial strain at a concentration of 2.5×10⁶ cfu/ml, and the bactericidal efficacies were shown in table 37.

TABLE 37 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B11 + + − − C11 + − − − B12 + + + − C12 + − − − + efficient, − inefficient

4) Salmonella: Salmonella was isolated from chick intestines in the chicken farm, the concentration of bacteria was 9.2×10⁴ cfu/ml, and the bactericidal efficacies were shown in table 38.

TABLE 38 Concentration (mg/ml) Polymer name 2.4000 1.2000 0.6000 0.3000 B11 + − − − C11 − − − − B12 + + + − C12 + − − − + efficient, − inefficient

EXAMPLE 6 Preparation of Poly (Acrolein, Acrylic Acid, Maleic Acid)

Invented drug (acrolein:acrylic acid:trans glutaconic acid (mol)=80:15:5): 29.20 g freshly distilled acrolein, 200.22 g freshly distilled acrylic acid and 20.16 g fumaric acid were added into a three-port flask with 800 ml absolute methanol, purged with nitrogen, 10.55 g benzoyl peroxide was added, and the solution was stirred and heated to reflux under nitrogen for 75 h. The reaction solution became a yellowish viscous liquid, which was dried under vacuum at 50-100° C. for 24 hours to form to Poly (acrolein, acrylic acid, maleic acid) with an average molecular weight no less than 1000.

EXAMPLE 7

The preparation of poly (acrolein, acrylic acid, cis-glutaconic acid) was the same as that in example 1.

EXAMPLE 8

The preparation of poly (acrolein, acrylic acid, and trans-glutaconic acid) was the same as that in example 1. 

1-10. (canceled)
 11. A terpolymer comprising monomers selected from the group consisting of:

wherein m and n are independently selected from integers equal to or greater than 1, and R is H, COOH or —R₁COOH, wherein R₁ is C₂-C₂₀ alkyl or alkenyl
 12. The terpolymer of claim 11, wherein R₁ is C₁-C₅ alkyl or alkenyl
 13. The terpolymer of claim 11, wherein R is —CH₂COOH.
 14. The terpolymer of claim 11, wherein the polymer has a molecular weight from about 1000 to about 10,000.
 15. The terpolymer of claim 11, wherein the monomers are derived from acrylic acid, acrolein, and alkyl alkenyl polybasic acid.
 16. The terpolymer of claim 11, wherein the monomers are derived from acrylic acid, acrolein, and fumaric acid.
 17. The terpolymer of claim 16, wherein the mole ratio of acrylic acid:acrolein:fumaric acid is 55-85:10-35:5-10
 18. The terpolymer of claim 16, wherein the mole ratio of acrylic acid:acrolein:fumaric acid is 63:30:7.
 19. A composition comprising the terpolymer of claim
 11. 20. The composition of claim 19, further comprising a pharmaceutically acceptable carrier.
 21. The composition of claim 19, wherein the composition is formulated as tablets, capsules, oral liquid, suspensions, pills, granules, ointment, or suppositories.
 22. A method of preparing the terpolymer of claim 11, comprising the steps of: (i) reacting acrylic acid, acrolein, and alkyl alkenyl polybasic acid with methanol; (ii) heating to reflux under nitrogen; and (iii) drying under vacuum to form the terpolymer.
 23. The method of claim 22, wherein the alkyl alkenyl polybasic acid is fumaric acid.
 24. The method of claim 23, wherein the mole ratio of acrylic acid:acrolein:fumaric acid is 55-85:10-35:5-10.
 25. The method of claim 23, wherein the mole ratio of acrylic acid:acrolein:fumaric acid is 63:30:7.
 26. A method of preventing or treating microbial infection in a subject, comprising the step of administering to the subject a composition comprising the terpolymer of claim
 11. 27. The method of claim 26, wherein the microbial infection is bacterial infection or parasitic infection.
 28. The method of claim 26, wherein the subject is an animal or a human. 